Polyploidy, mutation and hybridization with reference to medicinal plants. PHARMACOGNOSY & Phytochemistry-I (BP405T)Unit-IIPart-4 Polyploidy reference to medicinal plants. Types Of Polyploidy A. Euploidy a.Autopolyploidy b. Allopolyploidy B. Aneuploidy 1. Causes Of Polyploidy 2. Non-disjunction in mitosis 3. Non-reduction in meiosis 4. Polyspermy 5. Endo-replication or Endo- reduplication. Factors Promoting Polyploidy 1. Physical factor 2. Chemical factor 3. Biological factor Physical factor:- Temperature :- heat temperature & cold temperature Centrifugation X-rays Gamma rays Cosmic rays Ionizing & non-ionizing radiations UV-radiations Chemical factor:- Alkylating agents:- nitrogen & sulphur mustard Acridines Proflavins Nitrous acid Colchicines[6] Colchicines (Poisonous alkaloids):- Biological factor Mode of reproduction Mode of fertilization Breeding system present (Hybridization) Growth habit of the plant Size of chromosomes Application Of Polyploidy Mutation breeding Seedless fruits production Bridge crossing Ornamental & forage breeding Disease resistance through aneuploidy Industrial application of polyploidy mutation reference to medicinal plants Type of mutations: 1. Spontaneous and induced mutations. 2. Recessive and dominant mutations. 3. Somatic and germinal mutations. 4. Forward, back and suppressor mutation. 5. Chromosomal, genomic and point mutations Application Of Mutation: Hybridization reference to medicinal plants The following steps are involved in hybridization of plant: Choice Of Parents:. Selfing Of Parents Emasculation:. Bagging: Crossing Or Cross Pollination Labelling Collection Of Hybrid Seeds Significance of Hybridization

1. PHARMACOGNOSY & Phytochemistry-I
(BP405T)
Unit-II
Part-4
Polyploidy, mutation and hybridization with reference to
medicinal plants.
Name: Mrs. Pooja Deepak Bhandare
Assistant Professor
G H RAISONI UNIVERSITY
SCHOOL OF PHARMACY

2.  Polyploidy reference to medicinal plants.
• Polyploidy word is the combination of two words- „Poly‟+ „Ploidy‟, in
which Poly means many or more and Ploidy means the number of pairs or
sets of chromosomes available in cell of any living things. So polyploidy
defined as the condition at which the number of sets or pairs of
chromosomes will be more than two in cell of any living things. In which
one set inherited from each parents or excess of the diploid number

3. • Poly= many or more (may be 3,4,5,6......etc)&Ploidy= the number of
pairs or sets of chromosomes available in cell of any living things
(denoted by X or N), so Polyploidy= more than two sets or pairs of
chromosomes available in cell ofliving things[2].
• The number of sets or pairs of chromosomes (which is denoted by X or
N).Haploid (1n= gametes or chromatids) & diploid (2n) are normal form
of chromosomes but triploid (3n) & tetraploid (4n) are the example of
polyploidy (fig. 1).

4.  Types Of Polyploidy
• They are two types as follows:-
A. Euploidy
a) Autopolyploidy
b) Allopolyploidy
B. Aneuploidy

5. A. Euploidy: - Itis the types of polyploidy with multiple numbers of sets of
chromosomes which is complete in numbers to a specific species depending upon the
combination of the genomes. It is further classified in to 2 types:
a) Autopolyploidy:- Auto means self or same species. Polyploidy‟means the
multiplication the number of chromosomes. So the multiplication the number of
chromosomes between in same species is called “autopolyploidy”.
b) Allopolyploidy:- Allo means other or different species. Polyploidy means the
multiplication the number of chromosomes. So the multiplication the number of
chromosomes between in different species is called “allopolyploidy”

6. B. Aneuploidy:
Aneuploids are polyploids that contain either an addition or subtraction of one
or more specific chromosome(s) to the total number of chromosomes that
usually make up the ploidy of a species.
Term Chromosome Number
Monosomy 2n-1
Nullisomy 2n-2
Trisomy 2n+1
Tetrasomy 2n+2
Pentasomy 2n+3

7. Causes Of Polyploidy
• There are four types of causes behind the polyploidy:-
1. Non-disjunction in mitosis
2. Non-reduction in meiosis
3. Polyspermy
4. Endo-replication or Endo-reduplication

8. 1 Non-disjunction in mitosis:-
• As we know mitosis is the process of formation of two daughter cells from one parent cell.
• In the fig. 2 diploids (2n) has taken as an example for mitosis (generally found in early
embryoin cell), in which diploids are in the form of chromatids (single strand
chromosomes) then replicated and form copy of each chromatids and converted in to
double strand of chromosomes.
• Undergoes to separation of chromatids for cell division with formation of spindles but due
to disturbance in spindle protein, segregation of chromatids (non-disjunction) not occurs.
• Finally cell division not occurs.
• So the duplicated chromatids are present in same cell, ultimately the number of chromatids
will be increased. Result is that polyploidy occurs

9. Fig 2: Non-disjunction in mitosis

10. 2 Non-reduction in meiosis:-
• As we know meiosis is the process of formation of four daughter cells from
one parent cell.
• In the fig. 3 diploids (2n) has taken as an example for meiosis-1 (generally
found in early embryoin cell), in which 1diploids contains homologous
chromosomes and 1 diploids contains non-reduced chromosomes, then
replicated and form copy of each chromatids and converted in to double
strand of chromosomes.
• Undergoes to separation of chromatids for cell division with formation of
spindles but due to nondisjunctionit formsonly 2 daughter cellsin meiosis-1.
• But during meiosis-2 this daughter cells areform haploids & triploids which
further fertilized and form tetraploids (4n= 1n+ 3n)from haploids (1n) &
triploids (3n), which is the example of polyploidy.

12. 3. Polyspermy
• If the fertilization of egg (female nuclei) is done by more than one
male nuclei (in fig. 4), this condition is called Polyspermy.
• Which result is that triploid (example of polyploidy) is occurs. Very
rarely polyploidy is occurs due to polyspermy.

13. 4. Endo-replication or Endo-reduplication
• When the replication of DNA occurs in which the number of
chromosomes gets duplicated.
• But cytokinesis (cell-division) not occurs.
• Ultimately the increased numbers of chromosomes present in single cell.
• Result is polyploidy occurs during Endo-reduplication or Endo-
reduplication

14.  Factors Promoting Polyploidy
• There are three factors which promotes the polyploidy which are as
follows:
1. Physical factor
2. Chemical factor
3. Biological factor

15. 1 Physical factor:-
I. Temperature :- heat temperature & cold temperature
II. Centrifugation
III. X-rays
IV. Gamma rays
V. Cosmic rays
VI. Ionizing & non-ionizing radiations
VII.UV-radiations

16. 2. Chemical factor:-
I. Alkylating agents:- nitrogen & sulphur mustard
II. Acridines
III. Proflavins
IV. Nitrous acid
V. Colchicines[6]
VI. Colchicines (Poisonous alkaloids):-

17. Fig. 3.2 Normal Mitosis v/s Mitosis with Cholchicine

18. 3. Biological factor
I. Mode of reproduction
II. Mode of fertilization
III. Breeding system present (Hybridization)
IV. Growth habit of the plant
V. Size of chromosomes

19.  Application Of Polyploidy
• Mutation breeding
• Seedless fruits production
• Bridge crossing
• Ornamental & forage breeding
• Disease resistance through aneuploidy
• Industrial application of polyploidy

20. a) Mutation breeding
• High frequencies of chromosome mutations are desirable in modern breeding techniques,
such as tilling, as they provide new sources of variation. The multiallelic nature of loci in
polyploids has many advantages that are useful in breeding.
• The masking of deleterious alleles, that may arise from induced mutation, by their dominant
forms cushions polyploids from lethal conditions often associated with inbred diploid crops.
This concept has been instrumental in the evolution of polyploids during bottlenecks where
there is enforced inbreeding.
• Mutation breeding exploits the concept of gene redundancy and mutation tolerance in
polyploid crop improvement in two ways. First, polyploids are able to tolerate deleterious
allele modifications post-mutation, and secondly, they have increased mutation frequency
because of their large genomes resulting from duplicated condition of their genes.
• The high mutation frequencies observed with polyploids may be exploited when trying to
induce mutations in diploid cultivars that do not produce enough genetic variation after a
mutagenic treatment.
• This approach has been used in mutation breeding of Achimenes sp. (nut orchids) by first
forming autotetraploids through colchicine treatment followed by the application of fast
neutrons and X-rays. In this study, the autotetraploids were found to have 20-40 times higher
mutation frequency than the corresponding diploid cultivar due to the large genome

21. b) Seedless fruits
• The seedless trait of triploids has been desirable especially in fruits.
Commercial use of triploid fruits can be found in crops such as watermelons
and are produced artificially by first developing tetraploids which are then
crossed with diploid watermelon. In order to set fruit, the triploid
watermelon is crossed with a desirable diploid pollen donor

22. c)Bridge crossing
• Another breeding strategy that utilizes the reproductive superiority of polyploids
• is bridge crossing. When sexual incompatibilities between two species are due to
• ploidy levels, transitional crosses can be carried out followed by chromosome
• doubling to produce fertile bridge hybrids.
• This method has been used to breed for superior tall fescue grass (F.arundinacea) from Italian
ryegrass (2n=2x=14) and tall fescue (2n=6x=42) by
• using meadow grass (Fescue pratensis) as a bridge species. The same principle
• has been applied in fixing heterozygosity in hybrids by doubling the
• chromosomes in the superior progeny .

23. d) Ornamental and forage breeding
• One of the immediate and obvious consequences of polyploidy in plants is an
increase in cell size which in turn leads to enlarged plant organs, a phenomenon termed
gigas effect.
For example, the volume of tetraploid cells usually is about twice that of their diploid
progenitors. The increase in cell volume however is mainly attributed to increased
water and not biomass. Therefore, its application is limited for breeding agronomically
important crops such as cereals. Although chromosome doubling may result in
significantly larger seeds and increased seed-protein content in cereal crops, this
advantage is offset by low seed set

24. (e)Production of apomictic crops
• Most apomictic plants are polyploid but most polyploid plants are not
apomictic. In plants capable of both sexual and asexual reproduction,
polyploidy promotes the latter. Obligate apomicts are the most desired of
hybrids but little gain has been realized towards their development.
However, it has been suggested that obligate apomicts may be induced
through development of very high ploidy plants. An example of an obligate
apomict achieved at high ploidy level is the octoploid of the grass,
Themeda triandra.

25. f)Disease resistance through aneuploidy
• Aneuploidy has been applied in breeding to develop disease resistant plants
through the addition of an extra chromosome into the progeny genome. An
example is the transfer of leaf rust resistance to Tricum aestivum from
Aegilops umbellulata through backcrossing. In addition, other breeding
strategies utilizing aneuploidy have been explored including chromosome
deletion, chromosome substitution and supernumerary chromosomes.

26. g)Industrial applications of polyploidy
• Chromosome doubling is reported to have an apparent effect on many physiological properties
of a plant. The most discernable of these has been the increase in secondary as well as primary
metabolism.
• The resulting increase in secondary metabolites, in some cases by 100%, after chromosome
doubling has been widely exploited in the breeding of narcotic plants such as Cannabis, Datura
and Atropa. In vitro secondary metabolite production systems that exploit polyploidism have
also been developed.
• The production of the antimalarial sesquiterpene artemisinin has been enhanced six fold by
inducing tetraploids of the wild diploid Artemisia annua L. In addition, commercial synthesis of
sex hormones and corticosteroids has been improved significantly by artificial induction of
tetraploids from diploid Dioscorea zingiberensis, native to China.

27. • Attempts have been made to improve the production of pyrethrin, a
botanical insecticide, by chromosome doubling of Chrysanthemum
cinerariifolium. Other plants whose production of terpenes has increased
following artificial chromosome doubling include Carum cari, Ocimum
kilmandscharicum and Mentha arvensis. The enhanced production of
secondary metabolites such as alkaloids and terpenes in polyploids may
concurrently offer resistance to pests and pathogens

28. • Advantages Of Polyploidy:-
• Enlargement & increased vigour which is strength of the plant.
• Production of sterile triploids seedless fruits, generally by the help of
vegetative propagation except seedling & fertilization.
• Restoring fertility in hybrids.
• Overcoming barriers in hybridization.
• Enhancing pest resistance, disease resistance &stress tolerance plant

29. mutation reference to medicinal plants
• Sudden heritable change in the structure of a gene on chromosome or change the
chromosome number.
• Type of mutations:
1. Spontaneous and induced mutations.
2. Recessive and dominant mutations.
3. Somatic and germinal mutations.
4. Forward, back and suppressor mutation.
5. Chromosomal, genomic and point mutations

30. • Mutations can be artificially produced by certain agents called mutagens
or mutagenic agent. They are two types:
a. Physical mutagens:
(i) Ionizing radiations: X-rays, gamma radiation and cosmic rays.
(ii) Non-ionizing radiation: U.V. radiation,

31. b. Chemical mutagens:
(i) Alkylating and hydroxylating agents: Nitrogen and sulpher mustard; methyl and
ethylsulphonate, ethylethane sulphonates.
(ii) Nitrous acid:
(iii) Acridines: Acridines and proflavins. Ionizing radiation cause breaks in the
chromosome. These cells then show abnormal cell divisions. If these include gametes,
they may be abnormal and even die prematurely. Non-ionizing radiation like Ultra
Violet rays are easily absorbed by purine and pyrimidines. The changed bases are
known as photoproducts. U.V. rays cause two changes in pyrimidine to produce
pyrimidine hydrate and pyrimidine dimmers. Thymine dimer is a major mutagenic
effect of U.V. rays that disturbs DNA double helix and thus DNA replication.

32. Application Of Mutation:
• This method is rapid method and cheap for development of new variety.
• Induced mutagen are used for the induction of CMS (Congential Myasthenic
Syndrome)
Example. Ethidium bromide is used for induction of CMS in barley.
• It is effective for improvement of oligogenic character.
• This method is quik, simple and best way when a new character is to be induced.
• It improves the disease resistance in crop plant.
• It is used to improve the specific characters of well adapted high yielding varities.

33. Hybridization reference to medicinal plants
• It is mating or crossing of two genetically dissimilar plants having desired genes
or genotypes and bringing them together into one individual called hybrid. The
process through which hybrids are produced is called hybridization.
• Hybridization particularly between homozygous strains, which have been inbred
for a number of generations, introduces a degree of heterozygosis with resultant
hybrid vigour often manifest in the dimensions and other characteristic of the
plants. A hybrid is an organism which results from crossing of two species or
varieties differing at least in one set of characters.

34. • Based on the nature & relationship of plants to be crossed, hybridization
can be
1. Inter-varietal – Cross bet. plants of two diff. var. of samespecies - intra-
specific hybridization. Eg. Hybrid Maize.
2. Intra-varietal – Cross bet. two plants of diff. genotypes butsame variety.
3. Inter-specific – Cross bet. two species of genus - Eg. Wheat, Cotton,
Tobacco.
4. Inter-generic – Cross bet. two diff. genera. Eg. Sugarcane X Bamboo,
Wheat X Rye, Radish X Cabbage.

35. • The following steps are involved in hybridization of plant:
1. Choice Of Parents:.
2. Selfing Of Parents
3. Emasculation:.
4. Bagging:
5. Crossing Or Cross Pollination
6. Labelling
7. Collection Of Hybrid Seeds

36. 1. Choice of parents:.
First step in hybridization.
• Desirable male & female plants are selected.
• Parent plants should be healthy & vigorous.
• Parents to be grown in isolation &selfpollinated to bring homozygosity
in desirabletraits.
• All important characters to be combined shouldbe kept in mind.

37. 2. SELFING OF PARENTS
• 2 nd step in hybridization.
• Make the parents pure in its characters.
• Done by artificial self fertilization.
• Bisexual flowers - covered using paper bags beforeanthesis to prevent natural
cross pollination.
• Self pollination will occur inside the paper bags.
• In cross pollinated crops male & female flowers arebagged separately before
anthesis.
• When stigma becomes receptive, pollen grains arecollected & brushed on stigma
this is artificial pollination.
• Process continued till parent becomes homozygous or pure for its particular
character.

38. 3. EMASCULATION
• 3 rd step in hybridization.
• Removal of stamens from female parent beforethey burst & shed pollen. Done to prevent
self pollination.
• Done in flower buds expected to open in thefollowing day.
• Such flower buds are recognized by their enlarged unopened corolla. Emasculation can be
done by any of thefollowing methods
a. FORECEPS AND SCISSORS METHOD
• Done in large flowers.
• Buds are opened & anthers removed using sterile forceps & needles or scissors.
• No injury to be caused to other floral parts – calyx, corolla & especially pistil

39. b. HOT OR COLD WATER OR ALCOHOL TREATMENT
• For small flowers like that of rice, sorghum, etc. that are difficult to be emasculated by forceps,
scissors, etc.
• Emasculation is done by dipping panicles in hot water for a definite period.
• Ideal temperature is between 45o C and 50oC.
• Gynoecia can withstand a temperature at which anthers are killed.
• Also done by dipping inflorescence in cold water or alcohol for a definite period.
c. MALE STERILITY METHOD
• In self pollinated crops, emasculation is eliminated by the use of male sterile plants in which anthers
are sterile.
• Male sterility can be induced by spraying 2,4-D, NAA, maleic hydrazide, etc. on immature flower
buds.

40. 4. BAGGING
• After emasculation flower buds are kept enclosed in bags of ideal sizes, made of plastic, cellophane
or paper.
• Bags are tied by thread, wire, pins, etc.
• This process is called bagging.
• Both male & female flowers - bagged separately to prevent contamination (foreign pollen).
• Bagging is usually done before anthesis.
• MALE PARENT
• Unopened flower buds of male parent whether unisexual or bisexual, are covered as like female
parent to prevent contamination of foreign pollen grains.
• This is to ensure that pollen of bagged flowers are of the same plant and not of any other plant.

41. 5. CROSSING OR CROSS POLLINATION
• 5 th step in hybridization.
• It is defined as artificial cross pollination between genetically unlike parents.
• Viable pollen is collected from desired male plant & transferred on to the
stigma of the desired emasculated female parent.
• Pollen grains are collected in petri dishes.
• Bag is temporarily removed from the female parent & the collected pollen are
dusted on stigma.

42. • In crops like Bajara, Jowar, etc. hand cross pollination is tedious, since flowers are
small.
• In such crops male & female plants are grown side by side & male & female
inflorescences are enclosed in one bag. • Here crossing takes place inside the bag
automatically.
• Crossing is normally done at the time of anthesis, dehiscence & stigma
receptivity.
• Dehiscence of anther can be recognized by the yellowish powdery mass.
• Stigma receptivity is evident by the presence of fluid on its surface.
• After crossing female flowers are again bagged.

43. 6.LABELLING
• Emasculated & crossed flowers are properly bagged, tagged & labeled.
• Labeling is done either on bag itself or on separate labels.
• Labels are tagged to bags using thread.
• Label should bear following details –
Reference number
Date of emasculation
Date of crossing
Details of male and female parents

44. 7. COLLECTION OF HYBRID SEEDS
• After maturity of seeds, crossed heads of desirable characters are harvested &
collected along with labels in separate envelopes.
• In the next season, seeds of each head are sown separately to raise F1
generation.
• All plants of F1 are genetically similar & look exactly alike.
• Plants of F1 are progenies of crossed parents & are hybrids.
• They may exhibit hybrid vigor – increased growth, size, yield, function, etc.
over the parents.

45.  Significance of Hybridization
• The yield of crops can be increased by hybridization.
• By hybridizing different plants with better quality, a plant with superior quality can be produced.
• Lacking disease resistance is the main problem in high-yield crops: this problem can be solved
by producing disease-resistant plants by hybridization.
• By crossing the plants with different tolerance, a variety with high tolerance to stress can be
produced.
• Any desired characteristics like pest resistance, disease-resistant, and stress tolerance can be
attained by hybridization.
• The senescence period can be extended and aging can be reduced in some high economic crops.